Gene structure and promoter functional analysis of the marmoset type II GnRH receptor

The marmoset type II GnRH receptor (GnRH-R) gene has the same structure and genomic organisation as the human and other type II GnRH-R genes. The gene consists of three exons and two introns and overlaps in the antisense orientation on its 5' end with peroxisomal membrane protein 11beta and on its 3' end with the RNA-binding motif protein 8A. However, these genes occur only at one locus in the marmoset genome, while in the human at two loci. Employing 5' rapid amplification of cDNA ends demonstrated that the marmoset type II GnRH-R gene has two transcriptional start sites at -341 and -567 nucleotides relative to the translational start codon and both start sites lack TATA and CAAT consensus sequences. A luciferase reporter construct with a 2.3 kb 5' flanking region of the type II GnRH-R gene was active in a wide variety of cell lines tested, consistent with the wide tissue expression of the receptor. Progressive 5' and 3' deletions were employed to identify sequences required for basal expression of the type II GnRH-R gene. This analysis identified negative regulatory elements in the regions -2342/-1995, -1679/-1084 and -458/-1 and positive regulatory elements in the regions -1995/-1679, -1084/-458 and -458/-1 relative to the translational start site. The strongest of the positive regions located between -766/-665 has enhancer activity when cloned in front of a heterologous minimal promoter and is critical for basal expression of the type II GnRH-R.